Tank-binding Kinase-1 (TBK1) (pIC50 = 6.8)
GSK8612 suppresses type I IFN production in primary human mononuclear cells and toll-like receptor (TLR)3-induced IRF3 phosphorylation in Ramos cells. GSK8612 can prevent IFNβ from being secreted in THP1 cells in reaction to dsDNA and cGAMP, which is STING's natural ligand[1].

GSK8612 suppresses type I IFN production in primary human mononuclear cells and toll-like receptor (TLR)3-induced IRF3 phosphorylation in Ramos cells. GSK8612 can prevent IFNβ from being secreted in THP1 cells in reaction to dsDNA and cGAMP, which is STING's natural ligand[1].

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In a biochemical functional assay, GSK8612 inhibited recombinant TBK1 with an average pIC₅₀ of 6.8. [1]
In Ramos cells stimulated with the TLR3 ligand poly(I:C), GSK8612 inhibited the phosphorylation of interferon regulatory factor 3 (IRF3) at Ser396 with an average pIC₅₀ of 6.0, as measured by Western blot. [1]
In human peripheral blood mononuclear cells (PBMCs) stimulated with poly(I:C), GSK8612 inhibited the secretion of interferon-alpha (IFNα) with an average pIC₅₀ of 6.1, measured by a flow cytometry-based cytometric bead array (CBA) assay. [1]
In THP-1 cells stimulated with dsDNA-containing virus (Baculovirus), GSK8612 inhibited the secretion of interferon-beta (IFNβ) with a pIC₅₀ of 5.9. [1]
In THP-1 cells stimulated with the STING ligand cGAMP (60 µg/mL), GSK8612 inhibited the secretion of IFNβ with a pIC₅₀ of 6.3. [1]

Determination of inhibition of recombinant TBK1 [1]
\nThe inhibition of the enzymatic activity of TBK1 by GSK8612 was determined in duplicate by Reaction Biology Corporation using their Kinase HotSpot proprietary technology.\n[1]

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\n\nProtocol for affinity enrichment compound competition binding assay [1]
\nCompetition binding assays were performed as described previously by using Kinobeads and LipidKinobeads3,4 Briefly, 200 μL (1 mg protein) cell extract (see supplementary data) were pre-incubated with test compound or vehicle for 45 min at 4°C followed by incubation with kinobeads or lipid beads (7 μL beads per sample) for 1 hr at 4°C. The beads were washed with lysis buffer and eluted with 20 μL SDS sample buffer (250 mM Tris-HCl pH 7.4, 250 mM Tris Base, 20 % glycerol, 4 % SDS, 0.01 % Bromphenol blue). Proteins were digested according to a modified single pot solid-phase sample preparation (SP3) protocol. 5,6 Briefly, protein solutions were diluted to 2 % SDS and bound to S6 paramagnetic beads by addition of ethanol to a final concentration of 50%. Contaminants were removed by washing 4 times with 70% ethanol. Proteins were digested by resuspending in 0.1 mM HEPES (pH 8.5) containing TCEP, Chloracetamide, Trypsin and LysC following o/n incubation. TMT labeling was performed using the 10-plex TMT reagents, enabling relative quantification of 10 conditions in a single experiment. 4,7 The labeling reaction was performed in 40 mM triethylammoniumbicarbonate, pH 8.5, at 22°C and quenched with glycine. Labeled peptide extracts were combined to a single sample per experiment. Samples were dried in vacuo and resuspended in 0.05% trifluoroacetic acid in water. 30% of the sample was injected into an Ultimate3000 nanoRLSC coupled to a Q Exactive. Peptides were separated on custom-made 35 cm × 100 μm (ID) reversed-phase columns (ReproSil-pur C18-AQ, 1.9 µm) at 55°C. Gradient elution was performed from 2% acetonitrile to 40% acetonitrile in 0.1% formic acid and 3.5% DMSO over 3.3 h. Samples were online injected into Q-Exactive mass spectrometers operating with a data-dependent top 10 method. MS spectra were acquired by using 70.000 resolution and an ion target of 3E6. Higher energy collisional dissociation (HCD) scans were performed with 35% NCE at 35.000 resolution (at m/z 200), and the ion target settings was set to 2E5 so as to avoid coalescence. 7 The instruments were operated with Tune 2.9 and Xcalibur 4.1.\n[1]

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\n\nDetermination of physical chemical properties [1]
\nCLND solubility, CHROM LogD, Artificial membrane permeability and human serum albumin binding were all determined according to the procedures reported by Diaz et al.\n

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\n\nProtocol for determination of the unbound compound fraction in blood [1]
\nA rapid equilibrium dialysis technique using 48-well rapid equlibrium dialysis plates with LC-MS/MS analysis was used to determine the unbound fraction of GSK8612 in rat, mouse and human blood. Concentrations of 200 ng/ml and 1000 mg/ml of compounds were spiked into blood (0.5% final concentration of organic solvent). Phosphate buffered saline (100 mM sodium phosphate + 150 mM sodium chloride pH 6.9-7.2) was used as the dialysis buffer. 100 µL of spiked blood was allowed to equilbrate with 300 µL of dialysis buffer (n = 6) by incubation at 37oC for 4 h in rapid equilibrium dialysis plates. Samples were analysed by LC-MS/MS using Labetalol as the internal standard and the unbound compound fraction in buffer was calculated.\n

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\n\nProtocol for the determination of microsomal stability [1]
\nThe study was carried out by the CRO Cyprotex. Pooled mouse, rat or human liver microsomes (protein concentration 0.5 mg/mL) in phosphate buffer (pH 7.4) with NADPH (1 mM) were incubated with 0.5 µM of GSK8612 (0.25% DMSO) at 37°C for total of 45 minutes. Aliquots (50 µL) of each experiment were taken at 0, 5, 15, 30 and 45 min and the reactions were stopped by the addition of 100 µL methanol containing internal standard. These samples were centrifuged at 2,500 rpm for 20 min at 4°C to precipitate the proteins and the samples were then analysed by LC-MS/MS. Control samples were also run under same conditions but with no NADPH added. \n\n

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The biochemical TBK1 inhibition assay was performed using recombinant TBK1 kinase. The average pIC₅₀ of GSK8612 was determined to be 6.8. No further details on the assay protocol (e.g., substrate, detection method) were provided in the main text. [1]
The binding affinity (pK_d) and selectivity profile of GSK8612 against 285 kinases were determined using an affinity enrichment chemoproteomics approach with kinobeads. The kinobeads consist of immobilized kinase inhibitors that ligate the ATP binding site. Cell extracts from a mixture of HEK293, K-562, HepG2, and placenta cells were used for protein kinase profiling. Additionally, lipid kinase profiling was performed using a lipid kinase affinity matrix in mixed HeLa, Jurkat, and K-562 cell extracts. Compound binding was quantified by mass spectrometry to determine pK_d values. [1]

Inhibition of phosphorylation of IRF3 in Ramos cells [1]
Ramos cells (ATCC, 106 /well) were exposed to GSK8612 for 60 min in cell culture media (RPMI1640) containing 2% fetal bovine serum and then stimulated with poly(I:C) (30 µg/mL) for 120 min at 37°C, 5% CO2. Cells were then collected and washed once with ice-cold DPBS. The cells were lysed in 50 mM Tris-HCl (pH 7.4) containing 5% glycerol, 1.5 mM MgCl2, 20 mM NaCl, 1 mM Na3VO4, 0.8% (v/v) IGEPAL™-CA630, 50 nM Calyculin A, phosphatase inhibitors mix, protease inhibitors mix (aprotinin, bestatin, leupeptin, pepstatin, phosphoramidon), 25 mM NaF, 1 mM dithiothreitol. Cell debris were removed by centrifugation and the soluble proteins were denatured with NuPAGE™ LDS Sample Buffer supplemented with 50 mM dithiothreitol). The samples were analysis by Western blot using 4-10% polyacrylamide gel and antibodies. The relative phosphorylation of IRF3 was analysed in SDS lysates by Western blot. Band intensities were quantified using a LICOR ODYSSEY™ scanner. The normalised phospho-IRF3 signal is displayed as percent inhibition, with unstimulated cells giving the minimum signal and vehicle-treated poly(I:C)-stimulated cells giving the maximum signal. The pIC50 was derived from a four-parameter sigmoidal curve fit constrained to top (100%) using GraphPad Prism software (V7).

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Inhibition of IFNα secretion in human PBMCs [1]
Peripheral blood mononuclear cells (PBMC) were isolated from human whole blood by density centrifugation using a Ficoll-based medium). PBMCs were seeded in 96-well plates at 50,000 cells per well in 50 µL media (RPMI1640) with 10% fetal bovine serum. Cells were incubated with GSK8612 or vehicle (DMSO 0.2%) for 1 h at 37°C, 5% CO2. The cells were then stimulated with 100 μg/ml poly(I:C) for 16 h at 37°C, 5% CO2. The supernatant was collected and analysed by multiplex Cytometric Bead Array (CBA) Flex Sets for secreted IFNα. Percent inhibiton of IFNα secretion was calculated based on the mean fluoresence intensities (MFI) measured by flow cytometry.

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Inhibition of IFN [1]
The secretion in human THP-1 THP-1 cells (ATCC) were plated in 96-well tissue culture plates at 100,000 cells per well in 100 µL media (RPMI-140, 10 % heat-inactivated FBS, 1 % penicillin/streptomycin/amphotericin) followed by 45 min incubation at 37°C with increasing concentrations of GSK8612 (0.003-10 µM). Cells were stimulated with 10 µL Bacmam virus (7.32 X 108 pfu/mL) or 10 µL of 600 µg/mL cGAMP solution (in water). Levels of secreted IFN were measured from cell supernatants following a 20 hour incubation by electrochemiluminescence using a MESO Sector S 600 platform following the manufacturer’s instructions for the MSD 96-well MULTI-ARRAY Human IFN-b assay (K111ADB-2). Data from three technical replicate experiments were averaged, plotted as a function of GSK8612 concentration in Graphpad Prism 4.0 and fit to model of inhibition to determine the IC50.

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Cell culture and lysate preparation for chemoproteomic analysis [1]
HEK293 (ACC-305, DSMZ), 1 K-562 (ACC-10, DSMZ),2 HepG2 (ATCC), HeLa (ATCC), Jurkat (ATCC), and Ramos cell lines were maintained in standard cell culture media made of DMEM or RPMI-1640 supplemented with 10% fetal calf serum without antibiotics. Human placenta was sourced from Biopredic and ABS. Mixed HEK293, K-562, HepG2 and human placenta cell extracts and mixed HeLa, Jurkat, and K-562 cell extracts were prepared as described. 3 For the analysis of different kinase activation states, Ramos cells were either left untreated or were stimulated with 50 nM Calyculin A and 0.1% DMSO for 30 min at 37°C, 5% CO2, followied by lysis steps as described above for other lysates.

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For the IRF3 phosphorylation assay in Ramos cells: Ramos cells were stimulated with the TLR3 ligand poly(I:C). Cells were treated with GSK8612 at various concentrations. After stimulation, cells were lysed, and proteins were separated by SDS-PAGE. Phosphorylation of IRF3 at Ser396 and total IRF3 levels were detected by Western blot using specific antibodies. Band intensities were quantified by densitometry, and pIC₅₀ values were calculated based on the inhibition of pIRF3 normalized to total IRF3. [1]
For the IFNα secretion assay in human PBMCs: Human PBMCs were stimulated with poly(I:C) in the presence of varying concentrations of GSK8612. After 16 hours of incubation, cell culture supernatants were collected. The concentration of secreted IFNα was measured using a flow cytometry-based cytometric bead array (CBA) assay. The mean fluorescence intensity (MFI) data were normalized to vehicle-treated, stimulated controls to calculate percent inhibition and pIC₅₀ values. [1]
For the IFNβ secretion assay in THP-1 cells: THP-1 cells were stimulated either with dsDNA-containing baculovirus or with 60 µg/mL of the STING ligand cGAMP. Cells were co-treated with a range of concentrations of GSK8612. After incubation, supernatants were harvested. The amount of secreted IFNβ (in pg/mL) was quantified using a suitable immunoassay (assay type not specified in detail). Dose-response curves were generated to determine pIC₅₀ values. [1]

GSK8612 has a low calculated logD value, which translates to high aqueous solubility, exceeding its determined affinity for TBK1. [1]
GSK8612 exhibited low microsomal clearance in human and rat, and low to medium clearance in mouse. [1]
GSK8612 is highly protein-bound in mouse, rat, and human blood. [1]

[1]. Discovery of GSK8612, a Highly Selective and Potent TBK1 Inhibitor. ACS Med Chem Lett. 2019 Mar 11;10(5):780-785.

Serine/threonine protein kinase TBK1 (Tank-binding kinase-1) is a non-canonical member of the IkB kinase (IKK) family. This kinase regulates signaling pathways such as innate immunity, tumorigenesis, energy homeostasis, autophagy, and neuroinflammation. This article reports the discovery and characterization of a novel, highly efficient, and selective TBK1 inhibitor, GSK8612. In cell experiments, this small molecule inhibited Toll-like receptor (TLR)3-induced phosphorylation of interferon regulatory factor (IRF)3 in Ramos cells and the secretion of type I interferon (IFN) in primary human monocytes. In THP1 cells, GSK8612 inhibited dsDNA and cGAMP (the natural ligand of STING)-induced interferon β (IFNβ) secretion. GSK8612 is a small molecule TBK1 inhibitor with excellent selectivity, making it an ideal probe for in-depth investigation of the biological functions of TBK1 in immune, neuroinflammation, obesity, or cancer models. [1] In summary, the bioactivity of GSK8612 has been demonstrated, which can inhibit the phosphorylation of IRF3 in Ramos cells, the secretion of IFNα in human peripheral blood mononuclear cells (PBMCs), and the secretion of IFNβ in THP-1 cells at low micromolar concentrations. GSK8612 is a highly selective TBK1 inhibitor and is therefore an ideal tool for in-depth research on the physiological role of TBK1 in biological models such as immunity, neuroinflammation, obesity, and cancer. [1] GSK8612 is a 2,4-diaminopyrimidine derivative synthesized through two consecutive nucleophilic aromatic substitution reactions. [1] Molecular docking to the TBK1 crystal structure (PDB 4IWQ) predicts that GSK8612 binds to the ATP-binding pocket. Key interactions include hydrogen bonds between the pyrimidine core and the hinge region residue Cys89, and hydrogen bonds between the sulfonamide NH and the Asn140 and Asp157 side chains. [1]
Chemical proteomics analysis showed that in cell extracts, GSK8612 had a lower affinity for the phosphorylated (activated) form of TBK1 (pK_d 6.8) than for the inactivated form (pK_d 7.7). [1]
GSK8612 is considered a highly selective chemical probe that can be used to elucidate the biological functions of TBK1 in immune, neuroinflammatory, obesity, and cancer models. [1]

C17H17BRF3N7O2S

520.3268

519.029

C, 39.24; H, 3.29; Br, 15.36; F, 10.95; N, 18.84; O, 6.15; S, 6.16

2361659-62-1

137553174

White to off-white solid powder

1.7±0.1 g/cm3

671.0±65.0 °C at 760 mmHg

359.6±34.3 °C

0.0±2.1 mmHg at 25°C

1.674

1.77

3

11

7

31

688

0

FFPHMUIGESPOTK-UHFFFAOYSA-N

InChI=1S/C17H17BrF3N7O2S/c1-10-14(8-28(27-10)9-17(19,20)21)25-16-24-7-13(18)15(26-16)23-6-11-2-4-12(5-3-11)31(22,29)30/h2-5,7-8H,6,9H2,1H3,(H2,22,29,30)(H2,23,24,25,26)

4-((5-bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-ylamino)methyl)benzenesulfonamide

GSK-8612; GSK 8612; GSK8612; 2361659-62-1; 4-(((5-Bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)amino)methyl)benzenesulfonamide; CHEMBL4446892; 4-[[[5-bromo-2-[[3-methyl-1-(2,2,2-trifluoroethyl)pyrazol-4-yl]amino]pyrimidin-4-yl]amino]methyl]benzenesulfonamide; 4-((5-Bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-ylamino)methyl)benzenesulfonamide; GSK8612;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 125 mg/mL (~240.23 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)